Rejuvenation of BMSCs senescence by pharmacological enhancement of TFEB-mediated autophagy alleviates aged-related bone loss and extends lifespan in middle aged mice

Bone marrow stromal/stem cells (BMSCs) are generally considered as common progenitors for both osteoblasts and adipocytes in the bone marrow, but show preferential differentiation into adipocytes rather than osteoblasts under aging, thus leading to senile osteoporosis. Accumulated evidences indicate that rejuvenation of BMSCs by autophagic enhancement delays bone aging. Here we synthetized and demonstrated a novel autophagy activator, CXM102 that could induce autophagy in aged BMSCs, resulting in rejuvenation and preferential differentiation into osteoblasts of BMSCs. Furthermore, CXM102 significantly stimulated bone anabolism, reduced marrow adipocytes, and delayed bone loss in middle-age male mice. Mechanistically, CXM102 promoted transcription factor EB (TFEB) nuclear translocation and favored osteoblasts formation both in vitro and in vivo. Moreover, CXM102 decreased serum levels of inflammation and reduced organ fibrosis, leading to a prolonger lifespan in male mice. Our results indicated that CXM102 could be used as an autophagy inducer to rejuvenate BMSCs and shed new lights on strategies for senile osteoporosis and healthyspan improvement.


INTRODUCTION
Bone marrow stromal/stem cells (BMSCs) are generally considered as the common progenitors for both osteoblasts and adipocytes in bone marrow, and have been greatly shown for clinical application potential. 13][4] Thus, the balance between osteogenic and adipogenic lineage commitment of BMSCs is essential for bone homeostasis.][7][8][9][10][11][12][13] For example, modification of transcription factors, 6,7 epigenetics [10][11][12][13][14] and autophagy 5,15,16 that enhanced osteogenesis and decreased adipogenesis of BMSCs alleviated SOP in mice.The latest evidence uncovered that premature aging of skeletal stem/progenitor cells caused bone loss. 17Therefore, it is assumed that stimulation of bone formation by BMSCs rejuvenation in vivo is an effective and attractive strategy for age-associated bone loss. 18Although the only currently approved anabolic agent-teriparatide-a recombinant parathyroid hormone (rPTH), has been shown to stimulate BMSCs osteogenesis and suppress adipogenesis in a variety of manners, [19][20][21][22][23][24] several adverse factors, such as progressive blunting of bone formation after 18-24 months treatment, decreased osteoblast precursors and increased osteosarcoma risks, limit its widespread use. 18,23utophagy is a complex dynamic process involved in the degradation and correction of intracellular damaged proteins and organelles, playing a vital role in bone and other tissue homeostasis. 16,25,266][27][28][29] A previous study also demonstrated that suppression of autophagy in osteocytes resulted in skeletal aging. 30In the bone marrow, autophagy is required for BMSCs homeostasis and plays a fundamental role in the onset and progression of pathological osteoporosis. 26Decreased autophagy has been shown in aged BMSCs, while enhanced autophagy by rapamycin could direct osteogenic lineage of aged BMSCs and rescued bone loss. 31,32Therefore, targeting autophagy that can rejuvenate stem cells is one of the most promising strategies for geroprotection. 33][36] Hence, more safer autophagy agonists are needed to be explored.
Transcription factor EB (TFEB) is a key transcriptional regulator of autophagy and lysosomal biogenesis.Emerging discoveries demonstrated that TFEB overexpression promoted longevity and reduced the burden of diseases, holding great promise as a therapeutic strategy for multiple age-associated diseases. 37,38egulation of TFEB has been shown to control the activities of osteoblasts and osteoclasts, 39,40 the two main cells playing in the coupling of bone remodeling for homeostasis, implying its potential use in osteoporosis prevention.However, little is known about the relationship between TFEB activities and osteoporosis.As precursors of bone lineage cells, BMSCs directly contribute to bone remodeling by differentiating into osteoblasts, but how and to what extend TFEB regulates fate decision of aged BMSCs in bone marrow is still unclear.Beyond its photodynamic therapy, verteporfin also shows autophagy inhibition, 41 but little is known about the detailed effects on different steps involved in autophagy and fate decision of BMSCs.
In this study, we synthesized a novel small molecule compound (named "CXM102") that could promote autophagy activities in aged BMSCs via enhancement of TFEB nuclear translocation, leading to senescence rejuvenation and bone anabolic effects in middle age mice.Additionally, low dose and long-term administration of CXM102 showed better benefits for healthspan than rapamycin in mice, including extended lifespan, reduced serum levels of inflammation, less lipid droplets and fibrosis in organs.
Our results demonstrated that CXM102 could significantly counteract aberrant lineage allocations of aged BMSCs, alleviate osteoporotic bone loss, increase healthspan and longevity of middle age mice.

CXM102 induces autophagic influx in hBMSCs
As AMPK activators, arctigenin and its derivatives show remarkable therapeutic potential against a wide range of human diseases. 42,43But from an unsuccessful experience of synthesis of arctigenin derivatives, we tried to redesign and synthesize a novel compound (Figs.S1a and 1a, hydrogen spectrum in Fig. S1b).Then the bioactivities were screened by cell viability.CXM102 had no cytotoxic effects on several normal cells, including BMSCs, liver cells (L02), synoviocytes and skin fibroblasts (FEK4) (Fig. S1c), but exhibited significant inhibition of cell growth in many cancer cell lines (Hep3B, HCCLM3, HCT116, DU145, A549 and THP-1) and rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) in a dose-dependent manner (Fig. S1d), suggesting its bidirectional effects on cell growth in human normal cells and cancer cells.Given the roles of arctigenin in activation of AMPK pathway and mesenchymal stem cell fate regulation, 43,44 we tried to study the effects of CXM102 on autophagy activities in young and old hBMSCs by comparing with rapamycin.Interestingly, CXM102 significantly increased autophagic activities in both young and old hMSCs, as revealed by elevated levels of LC3II and decreased p62 (Fig. 1b, c).Considering that activation of autophagy can delay cellular senescence in BMSCs, 32 we analyzed the effect of CXM102 on autophagic flux in old hBMSCs treated with chloroquine (CQ).Compared with rapamycin, CXM102 increased higher autophagic influx in old hBMSCs (Fig. 1b-e).Additionally, autophagy related genes (Atg5, Atg7) and lysosome related genes (Lamp1, Ctsb, Ctsd) were also upregulated by CXM102, accompanied with the elevated expression of upstream regulatory gene Tfeb (Fig. 1f).These results demonstrated that CXM102 could increase autophagy in young and old hBMSCs.
CXM102 induces autolysosomes formation via TFEB nuclear translocation in aged hBMSCs Subsequently, we investigated the upstream regulators that control the autophagic activation by CXM102.As CXM102 could upregulate Tfeb and autophagy and lysosome related genes (Fig. 1f), we tried to investigate the protein level of TFEB, which has been considered as a master regulator of lysosomal biogenesis and autophagy. 38,48,49As expected, CXM102 upregulated total TFEB expression in aged hBMSCs (Fig. 3a).Given that the transcriptional activity of TFEB mainly depends on its cellular localization, we found that CXM102 remarkedly increased nuclear translocation of TFEB (Fig. 3b-d).To confirm the role of TFEB in CXM102-induced autophagy, we conducted siRNA and pharmacological inhibitors to explore the activities of TFEB.Transfection with siTFEB could downregulate Tfeb and its downstream genes expression within 24 h, but not in 72 h (Fig. S3a).This data was consistent with the results from immunoblotting and immunofluorescent experiments (Fig. S3b, c).These results suggested that siTFEB transfection failed to inhibit nuclear translocation of TFEB for long-term experiments.However, we screened an inhibitor, verteporfin (VP), for the special regulation of nuclear translocation of TFEB.VP didn't change the global gene and protein levels of TFEB (Fig. S3a, b), but inhibit its nuclear translocation (Fig. 3b-d) and downstream genes for 72 h (Fig. S3a).Interestingly, CXM102 partially rescued the VP-induced inhibition of TFEB translocation into nucleus (Fig. 3c, d).In accordance with the TFEB activities regulated by CXM102 and VP, markers for autophagosomes (LC3II) and lysosomes (LAMP1) exhibited similar expression (Fig. 3e-g).
Correspondingly, VP treatment led to deteriorated senescence of old hBMSCs, including higher SA-β-Gal activities, γH2AX and p16INK4a positive cells and lower MMP (Fig. S3d-f).These results demonstrated that CXM102 induced autophagy via TFEB nuclear translocation to suppress hBMSCs senescence.
CXM102 regulates lineage choice of hBMSCs through nuclear translocation of TFEB To explore the roles of TFEB in osteogenic and adipogenic commitment of hBMSCs regulated by CXM102, we induced aged hBMSCs to differentiation medium and detected the relationships between TFEB and specific transcription factors for osteogenesis and adipogenesis, Runx2 and PPARγ, respectively.When exposure to osteogenic induction, CXM102 increased osteogenesis of young hBMSCs revealed by ARS staining (Fig. 4a-i).At the molecular level, CXM102 promoted TFEB translocated into nucleus (Fig. S4a) and upregulated the gene and protein expression of Runx2 (Figs. 4b, S4b).However, VP inhibited the nuclear fraction of TFEB (Fig. S4a), downregulated both gene and protein levels of Runx2 (Figs. 4b, S4b) and decreased osteogenesis (Fig. 4a-i).Nevertheless, CXM102 could restore the VP-induced decline in osteogenesis.By contrast, CXM102 significantly induced TFEB translocation into nucleus (Fig. 4a-ii), but downregulated PPARγ expression (Figs. 4b and S4b) and inhibited adipogenesis of young hBMSCs while exposure to adipogenic induction (Fig. 4a-ii).Interestingly, VP also inhibited adipogenesis of young hBMSCs, and showed synergistic inhibition of adipogenesis with CXM102.These results suggested that CXM102 favored osteogenic differentiation of young hBMSCs.
The preferential shift of lineage commitment into adipocytes of aged BMSCs contributes to increased marrow adiposity and in turn impairs osteogenic regeneration and hematopoiesis.Recently, we have invented a new method that permitted both osteogenic and adipogenic differentiation of BMSCs in the same culture system. 50To better understand the effects of CXM102 on the fate determination of aged hBMSCs, we performed this bivalent differentiation assay to observed the final osteogenesis and adipogenesis.In control group, little mineralized nodules were formed as revealed by ARS staining, in which the lipid droplets were also distinguished (Fig. 4c, arrowheads).Meanwhile, more lipid droplets were detected by ORO staining (Fig. 4d), confirming the preferential differentiation into adipocytes of aged BMSCs.However, CXM102 remarkably reduced lipid droplets and increased mineralized nodules (Fig. 4c, d).Despite VP suppressed osteogenesis and promoted adipogenesis of aged hBMSCs, which was contrary to its effects on young hBMSCs, supplement of CXM102 reduced adipocytes and promoted osteogenesis.Furthermore, these results were further confirmed by gene and protein analysis of Runx2 and PPARγ (Fig. S4c, d), indicating that CXM102 could remarkably direct aged hBMSCs differentiation into osteoblasts at the expense of adipocytes.Taken together, these results suggested that CXM102 could remodel the bone-fat imbalance of aged hBMSCs in vitro.

CXM102 promotes bone formation and ameliorates age-related bone loss in mice
Considering the specifical osteogenesis of CXM102 controlled by VP, whether there were the same effects to regulate bone metabolism in vivo was still unknown.As bone mineral density (BMD) declined with ageing in all groups of mice, CXM102 significantly delayed the loss of BMD value compared with other groups, except for the young mice (Fig. 5a).After a 4-month-long treatment with the drugs,  5b).Conversely, serum CTX-1 increased with aging, but didn't significantly change upon CXM102 stimulation (Fig. 5c), suggesting that CXM102 may mainly regulate bone formation in vivo.Subsequently, we assessed the effects of CXM102 on bone microarchitecture of the distal femurs by micro-CT scanning.As shown in 2D and 3D images, CXM102 significantly reduced total bone loss compared with the old mice (Fig. 5d, e).Meanwhile, a significant increase of total bone volume (BV/TV), number (Tb.N) and thickness (Tb.Th) of trabecular bone, as well as a decrease of trabecular separation (Tb.Sp.) were presented in OC mice compared with the old mice (Fig. 5e).Furthermore, the maximum loading of bone fracture (Fig. 5f), stress (Fig. 5g) and Young's modulus (Fig. 5h) in CXM102-treated mice were also significantly increased compared with the old mice, suggesting that CXM102 could effectively improve bone strength and prevent fragility fracture of senile osteoporosis.
However, pretreatment with VP severely attenuated the bone anabolic effects of CXM102 in vivo, leading to lower BMD and serum P1NP, less bone mass and mechanical properties (Fig. 5).In addition, it was also important to note that long-term injection of VP may accelerate the bone loss and worsened senile osteoporosis, but the mechanisms remained unclear.CXM102 remodels the bone-fat imbalance of aged marrow stromal cells in aged mice Aged-related osteoporosis is featured with low bone mass and excessive accumulation of adipocytes in bone marrow.Whether the bone anabolic effects of CXM102 under aging were attributed to the lineage shift of aged BMSCs remained unclear.Next, we focused on the effects of CXM102 on bone marrow cells.In contrary to young mice, there were more adipocytes (Fig. 6a-ii, arrowheads) and less osteoblasts in old marrow, but CXM102 administration significantly increased osteoblasts and reduced adipocytes accumulation (Fig. 6a, e, f).These results were further confirmed by specific staining of Runx2 and perilipin (Fig. 6b, g, h).Then we immediately analyzed the effects of CXM102 on TFEB expression of bone marrow cells.Consistently, ageing led to decreased the nuclear expression of TFEB in marrow cells, while CXM102 dramatically increased the nuclear fraction in old mice (Fig. 6c, j).Meanwhile, CXM102 also elevated Runx2 level in marrow cells as revealed by colocalization staining with TFEB (Fig. S4a, c), suggesting the enhanced nuclear level of TFEB triggered by CXM102 promoted osteogenesis in old mice.Similar to the aged hBMSCs, pretreatment with VP blocked the bone-forming effects of CXM102 and reduced the nuclear TFEB positive stromal cells.
Recent studies have demonstrated that BMSCs expressing leptin receptor (LepR + ) were major source of bone and marrow adipocytes. 24,51,52To investigate if CXM102 could enhance the autophagy activities of LepR + BMSCs in vivo, we examined LepR and LC3II expression in the trabecular bone area of distal femur.Our results revealed that the double-positive cells in OC mice were significantly outnumbered by the old group, but was distinctly diminished by VP (Fig. 6d, k, l).These results indicated that CXM102 could promote autophagy activities of LepR + BMSCs in vivo, but was also inhibited by VP.
Notably, myelofibrosis was also observed in bone marrow of old mice (Fig. S5b, green arrowheads), and VP treatment exacerbated the myelofibrosis.To our surprise, CXM102 significantly suppressed the myelofibrosis in old mice (Fig. S5b, d).Consequently, these results indicated that CXM102 was not only able to remold bone-fat imbalance but also create a healthier environment for fate choice of aged marrow cells under aging.

CXM102 reduces serum inflammation and increases healthspan and longevity in male mice
Given that rejuvenation of BMSCs and autophagic promotion have shown great benefits for decreased inflammation and healthspan improvement, we next determined the long-term effects of CXM102 starting at middle age (14-month-old) in male C57BL/6 mice on aged-related inflammation and lifespan.As low-dose (8 mg/kg weight) of rapamycin is considered as gold standard for healthy aging, 34 we compared the long-term effects of the same dose of CXM102 on survival and health parameters with rapamycin (Rapa).The survival curves of control (DMSO), CXM102-and Rapa-treated mice began to separate at 3 months of administration (Fig. 7a).While rapamycin injection led to a 5.68% extension of median lifespan, CXM102 increased up to 22.3%.Chronic inflammation has been considered as a hallmark of aging.In this study, the serum levels of TNF-α, IL-1β and IL-6 in CXM102-treated mice were significantly lower than old and rapamycin-treated mice (Fig. 7b-d).However, our work also showed that rapamycin failed to reduce serum levels of IL-1β and IL-6 compared with control mice (Fig. 7c, d), despite it diminished serum level of TNF-α (Fig. 7b).This result suggested that CXM102 may had a better effect on inflammaging than rapamycin.
Aging is also characterized by multi-organ dysfunction, including but not limited to non-alcoholic fatty liver disease (NAFLD), renal fibrosis and myocardial fibrosis, and rapamycin has been shown to extend lifespan and healthspan effectively. 34,35In this work, we meanwhile compared the histomorphology changes of these main organs treated with DMSO, CXM102 or rapamycin.As fat droplets occurred in liver with aging, 6-month treatment with CXM102 diminished a larger number of fat accumulation than rapamycin did (Fig. 7e-i, f).While rapamycin had little and no significant impacts on renal fibrosis and diameter of renal tubules, CXM102 largely reduced renal fibrosis and increased the diameter of renal tubules, respectively (Fig. 7e-ii, g, h).In parallel to the changes occurred in kidney, heart fibrosis was also substantially decreased in CXM102-treated mice (Fig. 7e-iii and l).Taken together, these results suggested the more beneficial effects of CXM102 on healthy aging than rapamycin.

DISCUSSION
Autophagy plays fundamental roles in the homeostasis and fate determination of BMSCs, 15 and an age-related decline in autophagy is widely accepted as one hallmark of aging. 25,27,53mpaired autophagy resulted in adipogenic differentiation, 54 while increased autophagy led to osteogenic differentiation of BMSCs 32 and improve the therapeutic application of BMSCs. 55In this study, we provided a novel autophagy inducer, CXM102, showing better benefits than rapamycin, to rejuvenate aged hBMSCs through the enhanced nuclear translocation of TFEB.It is noteworthy that CXM102 had no effects on normal cell growth, but exhibited inhibitory effects on multiple cancer cells (Fig. 1d).As autophagy may act as a double-edged sword that both helps tissue homeostasis and facilitates cancer development, 28,56 further studies are required to explore the unknown roles of CXM102 in cancers.
TFEB is a master transcriptional regulator of lysosomal biogenesis and autophagy, but little is known about the relationship between TFEB activities and fate decision of aged hBMSCs.In this work, we found that total protein level of TFEB and the targeting genes were downregulated by siTFEB within 24 h, but restored to the initial levels at 72 h after transfection (Fig. S3a, b).This phenomenon could be explained by the immediate disruption of total tfeb transcription caused by siTFEB transfection, thus leading to downregulated TFEB level.But when cultured cells were proliferating, the dose of siTFEB was not enough to disrupt the RNA strands.Considering autophagy is an ongoing and dynamic process, it was not suitable for siRNA to perform long term experiments like differentiation.Fortunately, we found VP had a specific role in prevention of TFEB nuclear translocation (Fig. 3c).VP is an FDA-approved drug that normally used for photodynamic therapy in clinical, but it also inhibits autophagy without light activation. 41Nevertheless, little is known about the detailed effects involved in autophagy.Here we demonstrated that VP could inhibit nuclear translocation of TFEB to suppress the formation of autophagolysosome, decrease osteogenesis in aged hBMSCs.Interestingly, VP has been shown to attenuate heterotopic ossification by inhibiting osteogenesis of tendon stem cells via YAP/β-catenin pathway. 57Consistent with 3-MA (Fig. S2), VP also inhibited the cytoprotective effects of CXM102 on senescence of hBMSCs (Fig. S3d-f).Recent studies showed that the transcriptional activities of TFEB were mainly controlled by mTORC1 and AMPK kinases, 37,38,49,58 but how CXM102 regulated the intracellular sublocation of TFEB is still unclear.
Another hallmark of aged BMSCs is the skewed differentiation potency from osteoblast to adipocyte, 7 featured by increased lipid droplets formation and less mineralized nodules when exposure to differentiation medium.We found that CXM102 not only enhanced the nuclear translocation of TFEB, but also promoted Runx2 expression in nucleus in respond to osteogenic induction (Fig. 4a-i).Hence, there may be a synergetic effect of CXM102 with Runx2 to regulate the osteogenic differentiation of BMSCs.By contrast, CXM102 specifically inhibited PPARγ expression, thus leading to less lipid droplets formation (Fig. 4a-ii).Additionally, we conducted a bivalent differentiation system to observe the effects SOP is featured with increased marrow adipocytes, decreased BMD, bone mass and strength, leading to an increased risk of fractures. 59Here we demonstrated that CXM102 significantly delayed the loss of BMD and bone mass, but augmented bone strength (Fig. 5), suggesting its potentially use for osteoporosis.As the only tissue where bone and fat coexist in the same microenvironment, the bone marrow offers a unique window into the investigation of molecular events governing the lineage commitment of BMSCs. 6Considering that CXM102 promoted the nuclear translocation of TFEB, autophagy and preferential osteogenesis of aged hBMSCs in vitro, the in vivo antiosteoporosis effects could be reasonably explained by the lineage shift of aged BMSCs conducted by CXM102, which was consistent with reduced marrow adipocytes, increased osteoblasts and autophagy activities (Fig. 6).Besides, myelofibrosis is another challenge of aging.We found that myelofibrosis increased with age, but CXM102 significantly inhibited the myelofibrosis (Fig. S4b).Since LepR + BMSCs have been identified as key components of hematopoietic microenvironment 52,60,61 and responsible for myelofibrosis, 62,63 it was reasonable that enhancement of TFEB-mediated autophagy by CXM102 treatment in LepR + BMSCs could reduce the myelofibrosis, but further studies are needed to illustrate the detailed mechanisms.
In addition, we also found that long-term injection of VP accelerated bone loss and weaken the strength, accompanied with less osteoblasts, more adipocytes and osteoclasts, reduced nuclear TFEB activities in marrow cells, reminding that more attention should be paid for clinical use, despite CXM102 could partially alleviate the adverse effects (Figs. 5 and 6).Taken together, these results suggested that CXM102 could remodel a healthier osteogenic microenvironment in aged bone marrow for lineage allocation of BMSCs and hematopoiesis in vivo. 646][67] Accumulation of marrow adipocytes results in increased inflammation, and osteoclast formation and bone resorption by secreting (pro) inflammatory factors.Compared with control and rapamycintreated mice, our results revealed that CXM102 significantly reduced serum levels of (pro)inflammatory factors, including TNFα, IL-1β and IL-6 (Fig. 7b-d).Given that bone remodeling plays key roles in homeostasis of bone marrow microenvironment, 64 we proposed that the increased bone anabolism and attenuated serum inflammation may contribute to healthier hematopoiesis for bone and other organs that led to better survival in CXM102treated mice.Osteoimmunology is termed to study the interactions between skeletal and immune systems, [68][69][70] but whether CXM102 play roles in marrow inflammation and immune development is of great interest in future studies.
In summary, we clearly demonstrated the novel compound CXM102 could rejuvenate and remodel bone-fat imbalance of aged BMSCs via enhanced nuclear translocation of TFEB, leading to alleviated bone loss and increased healthspan and longevity in middle age male mice.Our results will shed new lights on therapeutic strategies for senile osteoporosis and healthy aging.

LIMITATION OF THIS STUDY
We acknowledge several limitations of this study.First, we did not determine the effects of CXM102 on female mice, mainly because of the possible perturbation of estrogen deficiency in female mice for senile osteoporosis.Second, only 4 and 5 mice were analyzed in the study of osteoporotic features and longevity, respectively.Despite the data within each group are relatively centralized, larger number of both male and female mice will be required to confirm the anti-osteoporosis effects of CXM102 in vivo.Third, whether the LepR + BMSCs are truly responsive to CXM102 and how the lineage choice changes in mice are unclear.Maybe more sophisticated experimental techniques like lineage-tracing models, will address these urgent issues.Finally, the posttranscriptional modification of TFEB has been extensively elucidated, yet it is important to uncover the mechanism that how CXM102 regulates the TFEB activities in subsequent studies.
Primary and secondary antibodies used in this study were shown in Table S1.Cytochemical staining dyes, including Alizarin red S (G8550) and Oil red O (O8010) were from Solarbio Inc.The working solution was prepared based on the manufacture's instruction.

Animal approval
All animal experiments were performed in accordance with the protocol approved by Guangxi University of Chinese Medicine Institutional Welfare and Ethical Committee (Approval No. DW2022).C57/BL6 mice were reared in a pathogen-free facility at a controlled temperature (23-25 °C), under a 12 h light and dark cycle with food and water provided ad libitum.The cage and bedding were changed once a week.3-month-old and 14-monthold mice were used in this study.

Effects of CXM102 on cell growth
To study the effects of CXM102 on cell growth, a density of 2000 cells per well of indicated cell types were seeded into a 96-well plate as described before. 71cells were treated with different concentrations of CXM102 (0, 5, 10, 20, 30, 50 μmol/L) for different time points (12, 24 or 36 h).Cell activities were determined by Cell Counting Kit-8 kit (Beyotime, C0038) following the manufacturer's instruction.

Effects of CXM102 on autophagy
To investigate the roles of autophagy and transcription factor EB (TFEB) in hBMSCs senescence, 2.5 mmol/L 3-MA or 50 μmol/L CQ was added into the culture medium for 4 h before CXM102 treatment.Then cells were fixed and detected by immunofluorescence (IF) or western blotting for the analysis of autophagic influx, autophagosomes, autolysosomes and TFEB activities as described below.
Quantitative real-time PCR Total cellular RNAs were isolated using RNA Extraction Kits (Solarbio, R1200) according to the manufacturer's instructions and quantified by Nanodrop One C (Thermo Fisher Scientific, Madison, USA). 1 μg RNA was analyzed by using a TaqMan One Step RT-qPCR Kit (Solarbio, T2210) on LightCycle 96 Instrument (Roche, Mannhein, Germany) according to the manufacturer's instructions.The primers used in this study are shown in Table S2.The relative expression of target genes was normalized to Gapdh and calculated using the 2 −ΔΔCT method.
Western blotting Total protein was extracted using a protein extraction kit (Solarbio, BC3710) and the protein concentration was determined by BCA (Beyotime, P0012) assay according to the manufacturer's instructions.Next, 25 μg of protein sample was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes.Then the membranes were blocked with 3% BSA in TBST buffer for 2 h at room temperature, followed by incubation with the indicated primary antibodies (Table S1) at 4 °C overnight and horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature.Targeted proteins were visualized with enhanced chemiluminescence solution (Beyotime, P0018S) and detected using a ChemiDoc Touch Imaging system (Bio-Rad, CA, USA).
Effects of CXM102 on differentiation induction Osteogenic, adipogenic, and bivalent differentiation and detection of BMSCs were strictly conducted as we described in a recent method article. 50imal experiments To study the effects of CXM102 on aged-related bone metabolism, 14-month-old mice were divided into four groups, followed by a period of 4-month-long treatment with DMSO (Indicated as "Old"), CXM102 alone (OC; 20 mg/kg body weight), verteporfin alone (OV; 10 mg/kg) and CXM102 combined with verteporfin (OVC).In OVC group, verteporfin was pretreated 2 weeks before CXM102 injection.All drugs were dissolved into 50 μL DMSO and injected subcutaneously twice a week.Mice were sacrificed 1 month after the final injection, serum and femurs were collected for analysis.

BMD measurement
To assess the effects of CXM102 on BMD changes in vivo, the BMD values of 14-month-old mice were measured as the initial time and then followed by a period of 4-month injection with DMSO (Marked as "Old"), CXM102 (OC), verteporfin (OV) alone or both (OVC).In addition, 3-month-old untreated mice (Marked as "Young") were used as positive control in this experiment.BMD was measured once a month by dual energy x-ray absorptiometry (DXA) as described by others. 74Briefly, mice were anesthetized with isoflurane (Yuyan Corporation, Shanghai, China) in a chamber, then were placed on the Lunar PIXImus densitometer platform (GE Medical-Lunar, Madison, WI, USA).Half of the distal femur was selected as area of interest and perform a scout scan.BMD value was recorded by the PIXImus software (Lunar Piximus 2.0).

MicroCT analysis
MicroCT analysis was performed using the settings according to the manufacture's instruction.Generally, Mouse femurs were dissected, fixed with 4% paraformaldehyde (Beyotime, P0099) for 48 h and stored in 70% ethanol, loaded into 10-mm diameter scanning tubes, and scanned with a peak tube voltage of 50 kV and current of 0.145 mA at 18 μm resolution, (Latheta LCT200, Hitachi Aloka, Japan).The 2D and 3D model visualization software (BeeViewer v3.4.1) and data analysis software (VGStudioMAX v2.2) were applied to analyze the distal femoral metaphyseal trabecular bone and the parameters of the diaphyseal cortical bone.We defined a region of interest (ROI) as the area between 2%-6% proximal to the growth plate in the distal femora.Cross-sectional images of the femora were established for 3D histomorphometry analysis of the trabecular bone.The percentage of bone volume (BV/TV), the number (Tb.N), thickness (Tb.Th) and space (Tb.Sp) of trabecular bone were analyzed.

Biomechanical testing
To investigate mechanical strength of femurs, an electronic universal material testing machine (AGS-10kNJ, Shimadzu, Japan) was used for the three-point bending test described in other's experiment. 31Before the test, the long and short diameters of the femoral shaft were measured with a Vernier caliper, then femurs were placed between two support abutments.The load was applied to middle of the femoral shaft through a touching probe.During this test, the posterior surface was in tension, and the anterior surface was in compression.The strength variables (bending modulus of elasticity, bending energy, maximum bending stress, and bending rigidity coefficient) were calculated with a standard engineering formula.

Lifespan analysis
To compared the effects of CXM102 and rapamycin on lifespan, middle-aged mice were divided into three groups, which were administered with rapamycin (8 mg/kg weight), CXM102 (8 mg/kg weight) or DMSO twice a week for a period of 6-month-long term. 1 month after the final injection, three mice from each group were sacrificed for assessment of histological changes of liver, kidney and heart.The other mice were freely fed until natural death.Survival curves were calculated by Origin 8.0 (OriginLab, Guangzhou, China).

Statistical analysis
Results are represented as means ± standard deviations.Statistical analysis was performed using Student's t-test as well as one-way analysis of variance (ANOVA) followed by the Tukey HSD test for post hoc comparison (Origin 8.0, OriginLab).Difference was considered significant when P < 0.05 indicated as *, while more significant when P < 0.01 indicated as **.

Fig. 1
Fig. 1 CXM102 increases autophagic influx in hBMSCs.a Structure of CXM102.Western blotting (b) and quantitative (c) analysis of autophagy influx in young and old hBMSCs stimulated by DMSO (Control), CXM102 or rapamycin with CQ or not.Represent images (d) and relative intensity (e) of LC3II puncta detection in young and old hBMSCs treated with DMSO (Control), CXM102, rapamycin or CQ.f Autophagy and lysosome related gene analysis in old BMSCs treated with DMSO (Control), CXM102 or rapamycin.Results are presented as means ± SD, n ≥ 3, *P < 0.05, ** P < 0.01

5Fig. 2 Fig. 3 Fig. 4
Fig. 2 CXM102 ameliorates the senescence-associated phenotypes of hBMSCs.Representative images (a) and quantitative analysis (b) of SAβ-gal and DAPI staining.Representative images (c) and quantitative analysis (d) of double immunofluorescent staining of p16INK4a and γH2AX.e Representative images and quantitative analysis of intracellular ROS level.f Representative images and quantitative analysis of relative gene levels of Runx2 and Pparγ for osteogenic and adipogenic differentiation with or without CXM102 treatment.Representative images (g) of JC-1 staining and quantitative analysis and intensity analysis of mitochondrial membrane potential (MMP) (h) in g. Results are presented as means ± SD, n ≥ 3. **P < 0.01

Fig. 5
Fig. 5 CXM102 ameliorates aged-related bone loss in male mice.a Representative curves of BMD value and the relative changes of BMD to the starting point followed by 4-month treatment with CXM102 (OC), verteporfin (OV), or both (OVC).Red asterisks indicate the comparing with the Old group, while blue asterisks indicate the comparing with OV group.Serum levels of P1NP (b) and CTX-1 (c) from 3-month (young), 14-month (old) and 18-month-old mice treated with CXM102 (OC), or verteporfin (OV), or both (OVC).d Representative 2D and 3D micro-CT images of the distal femoral trabecular bone microarchitecture.e Quantitative analysis of distal femoral parameters including bone volume to tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).Quantitative analysis of maximum loading (f) and maximum stiffiness (g) of femurs by a three-point bending assay.h Quantitative analysis of Young's modulus of femurs.Results are presented as means ± SD, n ≥3.*P < 0.05, **P < 0.01

Fig. 6
Fig. 6 CXM102 remodels the bone-fat imbalance of aged marrow stromal cells in middle aged mice.a Representative images of H&E staining for histological analysis of distal femurs from the indicated mice.(i-v) Enlarged areas from each section.b Double-staining of perilipin and Runx2 in bone marrow sections.c Immunohistologic analysis of TFEB in marrow cells.d Immunofluorescent double-staining of LepR and LC3 in bone marrow sections.Quantitative analysis of osteoblasts (e), adipocytes (f), Runx2 positive cells (g), perilipin positive cells (h), osteoclasts (i), nuclear TFEB positive cells (j), relative LepR positive cells (k) and LC3II intensity (l) to old mice.Results are presented as means ± SD, n = 5. *P < 0.05, **P < 0.01

Fig. 7
Fig. 7 Long-term treatment with CXM102 increases healthspan and lifespan in male mice.a Kaplan-Meier survival curve for mice treated either with DMSO, or rapamycin, or CXM102.Gray dashed lines indicated median lifespan.Statistic analysis of serum levels of TNF-α (b), IL-1β (c) and IL-6 (d) at 14-month (before injection) and 20-month (6-month after injection with DMSO, or rapamycin, or CXM102).e Representative images of histological analysis from liver (i, Oiled red O staining), kidney (ii, Masson's staining) and heart (iii, Masson's staining).Quantitative analysis of lipid droplets in liver (f), numbers of fibers in glomeruli (g) and diameter of renal tubules (h) in kidney and numbers of fibers (i) in heart.Results are presented as means ± SD, n = 5. *P < 0.05, **P < 0.01, n.s.means no significance